The long-term objective of this proposal is to develop "covalent tethering" technology and apply it to the drug target, caspase-3. Caspase-3 is one of a class of proteins that form the working machines of "programmed cell death" or apoptosis. Inhibitors of caspase-3 which block the downstream cascade and prevent cell death, would have significant therapeutic prospects in the diverse areas of stroke, myocardial infarction, spinal cord injury, sepsis, traumatic brain injury, arthritis, and Huntington's, Alzheimer's, and Parkinson's diseases. The proposed approach avoids the classic screening of numerous 'combinatorial' compounds, and allows for the rapid selection of fragments, which will subsequently be linked to construct inhibitors of protein action as drug leads. A set of caspase-3 mutants will be constructed which contain single mutations to cysteine introduced around the target site, and the mutants will be screened with compounds from a library of prepared sulfhydryl-containing components. Those compounds that bind in the site and react covalently with the introduced cysteine will be identified by mass spectrometry, visualized in the protein structure, and used to propose conjugated components for the next round of synthesis. The cognate un-tethered binding entities (that lack the sulfhydryl portion) will subsequently be linked to construct inhibitors of protein action as drug leads.